| Apple Valsa canker is a serious branch and trunk rot disease caused by the fungus Valsa spp.,which is common in apple orchards in our country and seriously affects the yield and quality of apples.In order to control diseases and minimize the amount of pesticides,biological control plays an increasingly important role in the fruit tree disease control system.Among them,bacillus is regarded as a promising biological control agent because of its characteristics of easy isolation and culture,obvious antagonism to pathogens,strong stress resistance and good colonization ability.Therefore,in this study,the phylogenetic status of Bacillus was clarified and its root surface colonization ability was analyzed based on the screening of bacillus strains with inhibitory activity against Apple Valsa pathogen.Through genome analysis,the related genes of bacteriostatic substances were predicted and their biocontrol potential was explored,in order to improve the safe and efficient biological control methods of apple putrefying disease and provide theoretical basis for the subsequent research and development of biological agents.In this study,Cytospora mali,a pathogen of apple Valsa canker,was successfully isolated and identified from the diseased apple leaf tissue.Then,bacillus was isolated and screened from soil and fruit tree leaves by soil suspension coating method and tissue culture method from typical orchards in Shaanxi Province.Phylogenetic trees were constructed according to the homology of ITS gene,and their genetic classification was identified.At the same time,the antagonistic rhizosphere and endophytic bacillus were screened by plate antagonistic culture method.Two dominant bacillus strains with biosafety and strong colonization ability were obtained from hemolysis safety,movement ability and biofilm formation ability tests.Then,the two strains of Bacillus were daubed on the main and side branches of apple tree through greenhouse pot simulation experiment,and the apple branches were infected by the pathogen of Cytospora mali.Finally,the control efficiency of the tested bacillus on the apple Valsa canker was evaluated by measuring the incidence,the length of the disease spot and the disease index.In addition,we also analyzed the genome of one strain of Bacillus velezensis Lw-6 with the best biocontrol efficiency,and found that Lw-6 has several genes involved in amino acid metabolism and secondary product synthesis,and can synthesize a variety of secondary metabolites that inhibit the growth of plant pathogens,providing reference for the further study of the biocontrol mechanism of Bacillus velezensis.The main results are as follows:1.A pathogen of apple Valsa canker CCNWWJF-020 was isolated by tissue isolation method.After morphological observation and ITS sequence analysis,a phylogenetic tree was constructed,and its taxonomic status was determined as the pathogen of apple Valsa canker Cytospora mali;2.291 strains of Bacillus were isolated and purified from rhizosphere soil and apple tree leaves in Qianyang,Xunyi,Baishui and Luochuan counties of Shaanxi Province.Through phylogenetic analysis of 16 S r RNA gene sequences,194 strains were identified as 33 species belonging to 7 genera.They are 1 species of Brevibacterium,Lysinibacillus,Natronbacillus,Alkalibacillus,Brevibacillus,Paenibacillus and 27 species of Bacillus respectively;3.Antagonistic Bacillus strains were screened by the five-point confrontation method.After primary screening,15 species of Bacillus were found to have better control efficiency on the apple Valsa canker,with the inhibition rate over 60% and the inhibition zone larger than 5 cm.Then through re-screening,6 strains of Bacillus have the highest inhibition rate which is more than 78% on Cytospora mali.The phylogenetic tree was constructed by further analysis of the six antagonistic Bacillus strains based on the sequence of rec A housekeeping gene.One strain was identified as Bacillus subtilis subsp.str.,and the other five strains were identified as Bacillus velezensis;4.The blood agar plate was used to evaluate the hemolytic safety of Bacillus,the semi-solid medium was used to characterize the motility of the strains,and the 96-well plate staining method and drying weighing method were used to detect the biofilm formation ability.Finally,two strains of Bacillus velezensis CCNWWJB-089 and CCNWWJB-090 were determined as the biological control strains for the subsequent verification test of apple Valsa canker control;5.Through greenhouse pot experiment,it was found that the two biocontrol agents could reduce the incidence of apple Valsa canker to a certain extent.The inhibition rate of CCNWWJB-089 to Valsa canker spot was 70.84%,and the inhibition rate of CCNWWJB-090 to Valsa canker spot was 66.32%;6.The complete genome of Bacillus velezensis Lw-6 was determined and annotated by GO,COG and KEGG databases.It was found that Lw-6 had a large number of genes involved in amino acid metabolism and secondary product synthesis.The anti SMASH tool was used to predict the gene clusters of 15 secondary metabolites of the bacterium,and it was found that the bacterium was capable of synthesizing a variety of secondary metabolites that inhibit plant pathogenic fungi,such as bacillaene,fengycin,bacillibactin,plipastatins and surfactin. |
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